Healing of wounds in skin and other epithelia involves a complex set of interactions between numerous components, including epithelial cells, peripheral nerves, and immune cells, as well as soluble and matrix molecules contributed by the various cell types (reviewed in Hom et al., Growth factor therapy to improve soft tissue healing. Facial Plast. Surg., 18:41–52, 2002). Dysfunctions associated with one or more of these components can lead to anatomical changes of intact skin and/or alterations in the ability of wounded epithelium to regain its normal histology and function. For example, sensory denervation of skin results in alterations in skin anatomy, including thinning of the epidermis, decreases in epidermal proliferation, and changes in the gene expression of Langerhans cells (Huang et al., Influence of cutaneous nerves on keratinocyte proliferation and epidermal thickness in mice. Neuroscience, 94:965–73, 1999; Hsieh et al., Epidermal denervation and its effects on keratinocytes and Langerhans cells. J. Neurocytol., 25:513–24, 1996; Li et al., Sensory and motor denervation influence epidermal thickness in rat foot glabrous skin. Exp. Neurol., 147:452–62, 1997; Laplante et al., Mechanisms of wound reepithelialization: hints from a tissue-engineered reconstructed skin to long-standing questions. FASEB J., 15:2377–89, 2001). Therefore, sensory denervation of skin can produce an underlying environment that is predisposed to the establishment of wounds that are refractory to healing.
There are several clinical examples, including diabetic and pressure ulcers, in which a similar sequence of events appears to play a significant role. The importance of dermal innervation was actually understood in ancient times, and described in detail in the Bible: leprosy, a disease of the peripheral nerves that leads to erosive, chronic skin lesions (Weinstein et al., Molecular mechanism of nerve infection in leprosy. Trends Microbiol., 7:185–86, 1999), is the first lesion for which this pattern was described.
Normal keratinocytes have a relatively high, age-dependent proliferative index. However, epidermal thickness is maintained at a relatively constant level throughout most of life, suggesting that an equilibrium exists among keratinocyte cell birth, squamae formation, and sloughing of the stratum corneum (Stanulis-Praeger and Gilchrest, Growth factor responsiveness declines during adulthood for human skin-derived cells. Mech. Ageing Dev.,35:185–98, 1986; Laplante et al., Mechanisms of wound reepithelialization: hints from a tissue-engineered reconstructed skin to long-standing questions. FASEB J., 15:2377–89, 2001). This equilibrium is highly influenced by innervation of the skin. Hsieh and colleagues have shown that, within seventy-two hours of denervation, there is a marked and significant thinning of the epidermis (Hsieh and Lin, Modulation of keratinocyte proliferation by skin innervation. J. Invest. Dermatol., 113:579–86, 1999), raising the possibility that denervation negatively affects the keratinocyte mitotic index. In the same study, Hsieh and colleagues examined BrdU incorporation in denervated rat skin. They noted that it was reduced to almost half of the incorporation on the contralateral side of the same animal, and that epidermal thickness was reduced by 70% within four days (Hsieh and Lin, Modulation of keratinocyte proliferation by skin innervation. J. Invest. Dermatol., 113:579–86, 1999). These data are consistent with a model in which denervation of the skin leads to a reduction in keratinocyte growth, resulting in epidermal thinning.
Importantly, the alterations in keratinocyte and Langerhans cell anatomy and functionality are reversible. By three months following mechanical nerve transection, axons have regenerated into denervated areas, and epidermal thickness has returned to baseline, as has the keratinocyte proliferative rate (Huang et al., Influence of cutaneous nerves on keratinocyte proliferation and epidermal thickness in mice. Neuroscience, 94:965–73, 1999).
One possible explanation for the above findings is that, in the absence of sensory innervation, there is a decrease in blood flow to the target field. Such an alteration may result in either the accumulation of inhibitory molecules—which would then negatively affect keratinocyte growth—or a reduction in the delivery of one or more growth factors to the dermal/epidermal boundaries—which might also reduce keratinocyte proliferation. This hypothesis is consistent with the known pathophysiology of diabetes, in which there is microvascular damage, peripheral neuropathy, and thinning of the skin, with a propensity toward the development of ulcers. However, while it is an attractive hypothesis, it lacks support in the literature.
For example, Monteiro-Riviere and colleagues have demonstrated in a number of species that there is no correlation between blood flow and epidermal thickness (Monteiro-Riviere et al., Interspecies and interregional analysis of the comparative histologic thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species. J. Invest. Dermatol., 95:582–86, 1990; Monteiro-Riviere et al., Laser Doppler measurements of cutaneous blood flow in ageing mice and rats. Toxicol. Lett., 57:329–38, 1991). Thus, the epidermal thinning that occurs after denervation appears to result from a direct inter-relationship between sensory fibers and keratinocytes. The foregoing observations also raise questions as to the perceived etiology of stasis ulcers. While the dogma holds that decubital ulcers are the result of poor blood flow and blood pooling in the skin, this has never been demonstrated or tested rigorously. Given the above-described data, it is possible that these ulcers develop as a result of pressure-induced peripheral nerve damage and subsequent thinning of the skin.
The findings discussed above do not address whether it is the axons per se, their associated Schwann cells, or both, that influence keratinocyte biology. However, consistent with other examples of nerve regeneration (Gondré et al., Accelerated nerve regeneration mediated by Schwann cells expressing a mutant form of the POU protein SCIP. J. Cell Biol., 141:493–501, 1998; Weinstein, D. E., The role of Schwann cells in neural regeneration. The Neuroscientist, 5:208–16, 1999; Weinstein et al., Molecular mechanism of nerve infection in leprosy. Trends Microbiol., 7:185–86, 1999; Strauch et al., The generation of an artificial nerve, and its use as a conduit for regeneration. J. Reconstr. Microsurg., 17:589–98, 2001), regenerating sensory fibers in the skin grow only in association with Schwann cells (Mihara, M., Regenerated cutaneous nerves in human epidermal and subepidermal regions. An electron microscopy study. Arch. Dermatol. Res., 276:115–22, 1984), suggesting that the Schwann cells play an integral role in wound healing. This possibility is further supported by the observation that glial growth factor (GGF) (also known as NDF and ARIA), which is secreted by Schwann cells following injury (Carroll et al., Expression of neuregulins and their putative receptors, ErbB2 and ErbB3, is induced during Wallerian degeneration. J. of Neurosci., 17:1642–59, 1997), stimulates keratinocyte proliferation and increases epidermal thickness (Danilenko et al., Neu differentiation factor upregulates epidermal migration and integrin expression in excisional wounds. J. Clin. Invest., 95:842–51, 1995).
Refractory skin lesions present a huge therapeutic challenge in patients with a range of underlying pathologies, including diabetic ulcers, venous stasis ulcers, pressure ulcers, burns, and trauma. The possibility of increasing the rate of surgical wound closure represents another, related, challenge, for such an increase would provide the benefit of limiting post-operative wound infection. Finally, great benefit would be derived from a means of enhancing reinnervation of healing skin, as this would likely limit paresthesias resulting from failed nerve regeneration. To meet some of these challenges, investigators have developed therapies that merely stimulate simple re-epithelialization. This approach, while offering some benefit, is limited, as epidermis will break down in the absence of repair of the underlying tissue. Accordingly, given the huge clinical implications associated with wound healing, there exists a need to develop a new, satisfactory therapy that will achieve more than mere stimulation of simple re-epithelialization.